Exploring the glycan repertoire of genetically modified mice by isolation and profiling of the major glycan classes and nano-NMR analysis of glycan mixtures.

The production of mice with genetic alterations in glycosyltransferases has highlighted the need to isolate and study complex mixtures of the major classes of oligosaccharides (glycans) from intact tissues. We have found that nano-NMR spectroscopy of whole mixtures of N- and O-glycans can complement HPLC profiling methods for elucidating structural details. Working toward obtaining such glycan mixtures from mouse tissues, we decided to develop an approach to isolate not only N- and O-glycans, but also to separate out glycosphingolipids, glycosaminoglycans and glycosylphosphatidylinositol anchors. We describe here a comprehensive Glycan Isolation Protocol that is based primarily upon the physicochemical characteristics of the molecules, and requires only commonly available reagents and equipment. Using radiolabeled internal tracers, we show that recovery of each major class of glycans is as good or better than with conventional approaches for isolating individual classes, and that cross-contamination is minimal. The recovered glycans are of sufficient purity to provide a "glycoprofile" of a cell type or tissue. We applied this approach to compare the N- and O-glycans from wild type mouse tissues with those from mice genetically deficient in glycosyltransferases. N- and O-glycan mixtures from organs of mice deficient in ST6Gal-I (CMP-Sia:Galbeta1-4GlcNAc alpha2-6 sialyltransferase) were studied by the nano-NMR spectroscopy approach, showing no detectable alpha2-6-linked sialic acids. Thus, ST6Gal-I is likely responsible for generating most or all of these residues in normal mice. Similar studies indicate that this linkage is very rare in ganglioside glycans, even in wild-type tissues. In mice deficient in GalNAcT-8 (UDP-GalNAc:polypeptide O-Ser/Thr GalNAc transferase 8), HPLC profiling indicates that O-glycans persist in the thymus in large amounts, without a major change in overall profile, suggesting that other enzymes can synthesize the GalNAc-O-Ser/Thr linkage in this tissue. These results demonstrate the applicability of nano-NMR spectroscopy to complex glycan mixtures, as well as the versatility of the Glycan Isolation Protocol, which makes possible the concurrent examination of multiple glycan classes from intact vertebrate tissues.

Exploring the outcome of genetic modifications of glycosylation in cultured cell lines by concurrent isolation of the major classes of vertebrate glycans.

In the preceding article (Manzi,A.E., Norgard-Sumnicht,K., Argade,S., Marth,J.D., van Halbeek,H. and Varki.A. [2000] GLYCOBIOLOGY:, 10, 669-688), we reported a comprehensive approach for the extraction, fractionation, and isolation of all of the major classes of sugar chains (glycans) from vertebrate tissues. Here we apply this "Glycan Isolation Protocol" to a variety of cultured mammalian cell lines, including two wild-type Chinese hamster ovary (CHO) cell lines and some of their genetically modified variants that were predicted or known to have defined abnormalities in the biosynthesis of one or more classes of glycans. We also use this approach to characterize clone 489, a new derivative of the GAG-deficient CHO clone pgsA-745, in which sulfation has been restored by transfection of a wild-type CHO cDNA library. By metabolically labeling the cell lines with [6-(3)H]glucosamine we were able to monitor the recovery of all major classes of glycans. The results allow us to reach several conclusions: first, the protocol described in the preceding paper is further validated by finding good recovery of total radioactivity and appropriate distribution of label in the correct glycan classes in the fractions from a variety of cell lines; second, the amount of radioactivity recovered in free glycosylphosphatidylinositol (GPI) lipids is remarkably high when compared to that found in GPI anchors, with the former being the dominant form in some cells; third, cells with known genetic mutations in specific glycosylation pathways are shown to have the expected changes in the distribution of recovered radioactivity in the appropriate fractions; fourth, the N- and O- glycans recovered via the protocol are of adequate quality to demonstrate marked differences in their structural profiles and/or content; fifth, the protocol can pick up unexpected differences of glycan classes not predicted to be affected by the primary defect; finally, the reappearance of sulfation in the novel clone 489 is not due to restoration of GAG sulfation, but rather due to the new expression of sulfation in the fraction enriched in N- and O-linked glycopeptides. These results demonstrate the power of this comprehensive approach for the concurrent exploration and profiling of the different major classes of glycans in cells.

Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents.

The selectins are calcium-dependent C-type lectins that bind certain sialylated, fucosylated, sulfated glycoprotein ligands. L-selectin also recognizes endothelial proteoglycans in a calcium-dependent manner, via heparan sulfate (HS) glycosaminoglycan chains enriched in unsubstituted glucosamine units. We now show that these HS chains can also bind P-selectin, but not E-selectin. However, while L-selectin binding requires micromolar levels of free calcium, P-selectin recognition is largely divalent cation-independent. Despite this, HS chains bound to P-selectin are eluted by ethylenediamine tetraacetic acid (EDTA), but only at high concentrations. Porcine intestinal mucosal (mast cell-derived) heparin (PIM-heparin) shows similar properties, with no binding to E-selectin, calcium-dependent binding of a subfraction to L-selectin and to P-selectin, and calcium-independent binding of a larger fraction to P-selectin, the latter being disrupted by high EDTA concentrations. Analysis of defined heparin fragment pools shows a size dependence for interaction, with tetradecasaccharides showing easily detectable binding to L- and P-selectin affinity columns. L-selectin binding fragments include more heavily sulfated and epimerized regions and, as with the endothelial HS chains, they are enriched in free amino groups. The P-selectin binding component includes this fraction as well as some less highly modified regions. Thus, endothelium-derived HS chains and mast cell-derived heparins could play a role in modulating the biology of selectins in vivo. Notably, P- and L-selectin binding to sialyl-Lewisx and to HL-60 cells (which are known to carry the native ligand PSGL-1) is inhibited by unfractionated pharmaceutical heparin preparations at concentrations 12-50-fold lower than those recommended for effective anticoagulation in vivo. In contrast, two low molecular weight heparins currently considered as clinical replacements for unfractionated heparin are much poorer inhibitors. Thus, patients undergoing heparin therapy for other reasons may be experiencing clinically significant inhibition of L- and P-selectin function, and the current switchover to low-molecular weight heparins may come at some loss of this effect. Low-dose unfractionated heparin should be investigated as a treatment option for acute and chronic diseases in which P- and L-selectin play pathological roles.

Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1.

Selectins interact with glycoconjugate ligands in important normal and pathological situations. While high affinity recognition of natural ligands is associated with alpha 1-3(4)fucosylated, alpha 2-3sialylated (and/or sulfated) lactosamine sequences, small oligosaccharides that potently inhibit the selectins have not been found. One possibility suggested by other investigators is that high affinity may require unusual sequences not yet tested, for example, the "major capping group" (6'-sulfo-sialyl Le(x)) of the L-selectin ligand GlyCAM-1. To explore this possibility, we synthesized a spectrum of novel synthetic and semisynthetic oligosaccharides related to those on natural ligands. In studying these molecules, we noted that binding of recombinant soluble selectins to immobilized sialyl Le(a) or 3'-sulfo-Le(x) is markedly inhibited by concentrations of chloride above the physiological range. This indicates the ionic nature of the interactions, and shows that buffers typically used in screening assays for inhibitors are not optimal. Using parameters that more closely approximate physiological conditions, we confirmed that alpha 2-3-linked sialic acids, and alpha 1-3(4)fucosylation are important for recognition. Similar results obtained with both types of immobilized targets for the three selectins indicated that the binding sites for sialic acid and sulfate are very close, or identical. While O-sulfate esters mostly improved L- and P-selectin recognition, effects depended upon their position and number. Furthermore, sulfation can also impart some "negative" specificity: the major capping group does not interact with E-selectin. The branched Core 2 sequence seemed to enhance L- and P-selectin binding, however, the best inhibitors still appeared to be sialyl Le(a) and 3'-sulfo-Le(x), with the aglycone group of the latter affecting binding. Of particular note, the "major capping group" of GlyCAM-1 was not an unusually potent nor highly selective inhibitor of L-selectin, even when studying the interaction of L-selectin with native GlyCAM-1 itself.

Unusual anionic N-linked oligosaccharides from bovine lung.

We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups.

We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L-selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L-selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L-selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed.

Calcium-dependent heparin-like ligands for L-selectin in nonlymphoid endothelial cells.

L-Selectin is a calcium-dependent mammalian lectin that mediates lymphocyte trafficking by recognizing sialylated ligands on high endothelial venules in lymph nodes. Although L-selectin probably mediates neutrophil extravasation into nonlymphoid tissues, no corresponding ligand has been characterized. Staining of cultured endothelial cells with an L-selectin chimera (LS-Rg) showed an internal pool of ligands. Metabolic labeling with sulfur-35-labeled sulfate revealed heparin lyase-sensitive ligands that bound LS-Rg in a calcium-dependent, sialic acid-independent manner. A fraction of commercial heparin bound to LS-Rg and LS-Rg bound to heparin-agarose, both in a calcium-dependent manner. Thus, L-selectin recognizes endothelial heparin-like chains, which could be physiological ligands mediating leucocyte trafficking.